Present approaches are unable to cure rheumatoid arthritis (RA) and other chronic autoimmune diseases. The current proposal plans to develop a novel approach that tests the central hypothesis: CD4?? regulatory T cells (iTregs) generated with IL-2 and TGF-2 are stable in an inflammatory milieu and are able to treat collagen-induced arthritis (CIA). While thymus- derived, naturally-occurring CD4???? (nTregs) suppress Th1- or Th2-cell-mediated autoimmune diseases, these cells are less successful in controlling (IL-17-producing) Th17 cell- mediated diseases such as CIA. Additionally, unlike iTregs, nTregs have increased cell plasticity, and can be converted into Th1, Th2 or Th17 cells while losing their suppressive activities in the presence of pro-inflammatory cytokines. We and others have established TGF-2 is able to convert naove CD4? cells to iTregs that share similar phenotypic and functional characteristics with nTregs. Interestingly, our recent studies revealed that unlike nTregs, iTregs did not make conversion to Th17 and Th1 cells in the presence of pro-inflammatory cytokines. In addition, iTregs but not nTregs maintained the suppressive activity against T cell response in the presence of IL-6 in vitro. Moreover, iTregs but not nTregs even prevented other T cells from becoming Th17 cells in the presence of IL-6 and TGF-2. However, pretreated nTregs with IL- 2/TGF-2 or atRA alters the plasticity and restore functionality of nTregs. Based on these preliminary data, we anticipate that iTregs and pretreated nTregs are stable following adoptive transfer into the established autoimmune arthritis. We expect iTregs and pretreated nTregs can significantly ameliorate the clinical signs of the established arthritis following treatment. We also expect that combination of all-trans retinoic acid (atRA) and TGF-2, or antigen-specific iTregs can enhance the therapeutic effects of iTregs on the established arthritis. We believe that iTregs not only directly suppress T cell response, but also induce the formation of tolerogenic DCs and these DCs produce IL-27, atRA and/or IDO that eventually restrain Th17 cell differentiation and function. Accordingly, the project has three specific aims: 1) Determine the relative stability of nTreg and iTreg cells when adoptively transferred into established collagen-induced arthritis (CIA). nTreg or iTregs will be sorted or induced from DBA/1 Foxp3-GFP knock-in mice or Foxp3-GFP/IL-17-RFP double knock-in mice. nTreg or iTregs cells will be adoptively transferred into DBA/1 or C57BL/6 mice at day 14 or day 28 after immunization with collagen II (CII) and Complete Freund's Adjuvant (CFA). The migration, distribution, survival, phenotype (Foxp3) and conversion into T help cells (Th1, Th2 or Th17 cells) of Treg subsets will be monitored with GFP and RFP expression. 2) Compare the therapeutic effect of both nTreg and iTreg cells on development of collagen-induced arthritis. nTreg, iTreg or control cells will be administrated to DBA/1J mice on day 14 or day 28 after immunization with CII/CFA. The protective effect of these cells will be judged by arthritis incidence and severity, levels of anti-CII IgG2a antibodies in sera and histological examination of arthritic limbs. 3) Define the cellular and molecular mechanism(s) by which iTregs are resistant to Th17 cell conversion and regulate Th17 cell differentiation and function in the inflammatory milieu. We will examine whether the transcription factor T-bet and Th1 cytokine expression in iTregs are responsible for their resistance. We will also examine whether these factors or tolerogenic DCs induced by iTregs contribute to suppressing Th17 differentiation and function. The results from this study will promote the understanding of therapeutic effects of Treg cells in the prevention and cure of rheumatoid arthritis. If successful, this project will also have a direct clinical relevance and possibly provide a novel approach to treat RA and other autoimmune diseases which may not have the severe side effect(s) characteristic of current therapies.